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Link to original content: https://pubmed.ncbi.nlm.nih.gov/27903072
A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo - PubMed Skip to main page content
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. 2017 Jan;28(1):125-134.
doi: 10.1089/hum.2016.140.

A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

Jiamiao Lu  1   2 , James A Williams  3 , Jeremy Luke  3 , Feijie Zhang  1   2 , Kirk Chu  1   2 , Mark A Kay  1   2
Affiliations

A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

Jiamiao Lu et al. Hum Gene Ther. 2017 Jan.

Abstract

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.

Keywords: AAV vectors; enhance transgene expression; intron; mini-intronic plasmid; miniorigins.

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Conflict of interest statement

Author Disclosure James Williams and Jeremy Luke have an equity interest in Nature Technology Corporation. Jiamiao Lu, Feijie Zhang, Kirk Chu, and Mark Kay declare that no other competing financial interests exist.

Figures

<b>Figure 1.</b>
Figure 1.
RSV-hAAT expression, recombinant adeno-associated virus (AAV) vectors, and transgene expression in mice. (a) Schematic of OIPR intron and recombinant AAV vectors. (A) Structure and sequence of OIPR intron; 5′-exon splicing enhancer (5′-ESE), G triplets, branch point sequence (BPS), 11 nucleotide polypyrimidine track, and 3′-ESE are incorporated for efficient splicing (underlined). Arrows indicate the sequence where splicing takes place. (B) Schematics of recombinant AAV vectors with (AAV.OIPR.RHB) and without (AAV.RHB) OIPR intron. (b) Serum hAAT levels at various time points after infusion of the corresponding recombinant AAV8 (rAAV8) vectors (n = 5/group, 1.0 × 1011 VG/mouse). Error bars represent the standard deviation (SD) for all of the figures.
<b>Figure 2.</b>
Figure 2.
RSV-hAAT expression constructs and transgene expression in mice. (a) Schematic of plasmid, minicircle, and mini-intronic plasmid (MIP) vectors with full-length or truncated OIPR intron. Serum hAAT levels at various time points after equimolar infusion of each vector were determined (n = 5/group). (b) Schematic of MIP vectors with full-length and 1 kb OIPR. Serum hAAT samples were determined at various timepoints after equimolar infusion of each vector (n = 5/group). (c) Schematic of plasmid, minicircle, and MIP vectors with full-length and 0.5 kb OIPR. ELISA results by analyzing serum samples at various time points of animals infused with equimolar amounts of each vector (n = 5/group). Error bars represent SD.
<b>Figure 3.</b>
Figure 3.
R6K and ColE2 miniorigins enhanced transgene expression. (a) Schematic of plasmid and MIP vectors that utilize miniorigins R6K-RNA-OUT or ColE2-RNA-OUT as plasmid backbone or promoter proximal introns. Serum hAAT levels at various time points after hydrodynamic infusion of equimolar amounts of the plasmids, respectively, were determined (n = 5/group). (b) Schematic of minicircle and MIP vectors that were tested in mice for the abilities of miniorigin introns to enhance transgene expression in MIP vectors. Serum hAAT levels at various time points after hydrodynamic infusion (n = 5/group). (c) Schematic of plasmid, minicircle, and MIP vectors. Serum samples at various time points from mice hydrodynamically infused with equimolar amounts of these vectors were analyzed for hAAT expression using ELISA (n = 5/group). Error bars represent SD.
<b>Figure 4.</b>
Figure 4.
R6K and ColE2 miniorigins were able to enhance transgene expression from AAV when used as 5′ promoter proximal introns. (a) Schematic structure of recombinant RSV promoter AAV vectors with or without OIPR/R6K/ColE2 introns. Serum hAAT levels at various timepoints after administration of rAAV8 (1.0E11 VG/mouse) (n = 5/group). (b) Schematic structure of recombinant ApoE promoter AAV vectors with human factor 9 (hFIX) intron 1/OIPR/R6K introns. rAAV8 vectors (1.0 × 1011 VG/mouse) were administered and serum hAAT levels determined at various timepoints (n = 5/group). (c) Schematic structure of recombinant phosphoglycerate kinase (PGK) promoter AAV vector DNAs with or without introns. The vector plasmid DNAs were infused into mice at equimolar amounts via hydrodynamic tail vein injection. Serum hAAT samples were determined at various time points (n = 5/group). Error bars represent SD.
<b>Figure 5.</b>
Figure 5.
Incorporating of the miniorigin introns does not change rAAV vector copy number in vivo. Liver DNA samples of animals infused with different rAAVs from experiment shown in Fig. 4a were extracted at 77 days after injection. Copy number of each vector per diploid genome in these liver samples was determined by quantitative real-time PCR. Two duplicate samples from each animal and two technical repeats for each sample were tested. The standard deviation was determined by using four quantitaive PCR reactions of animal samples from each infusion group.
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